The overall objective of the proposed research is to characterize the molecular mechanisms involved in the tissue-specific expression of the human apolipoprotein (apo) E/C-I and apoA-I/ C-III/A-IV gene complexes. Initially, the regulatory elements that determine expression in specific tissues will be localized to short segments of genomic DNA by preparing transgenic mice with various constructs of the apolipoprotein genes. When the sequences that contain these elements have been identified, they will be characterized through the use of cultured cell transfections and by cell-free transcription using appropriate constructs. The nuclear proteins that bind to these control sequences will be identified by DNase I footprinting and gel mobility retardation, and they will be isolated by oligonucleotide affinity chromatography and related methods. These proteins will be tested for their influence on transcription rates by using cell-free transcription assays. If these factors are proteins that have not been isolated and characterized for other genes, then the cDNAs that encode the apolipoprotein gene transcription factors will be isolated and their nucleotide sequences will be determined. Their relationship to other transcription factors will be investigated. The cell types that express the apolipoprotein genes will be identified by in situ hybridization and immunocytochemistry. This cell-specific gene expression will be investigated in the adult as well as during fetal and neonatal development to determine if there are any variations in the activities of the tissue-specific elements. The identification and action of the individual control sequences will be verified via transgenic mice prepared with constructs designed to test the function of the regulatory sequences, and capable of specifying expression in single cell types. This knowledge will be used to understand the physiological roles of these apolipoprotein genes. In particular, the apoE/C-I gene complex has a unique pattern of tissue distribution that appears to be linked to local metabolic requirements, and abnormal alterations in its expression are associated with disorders of lipid metabolism and the development of atherosclerosis.